The acute regulation of steroid hormone production in steroidogenic tissues is controlled by the delivery of the substrate cholesterol to the inner mitochondrial membrane which is the site of the cholesterol side chain cleavage enzyme system (CSCC). This transfer occurs as a result of the de novo synthesis of a protein(s) whose existence has been known for greater that 30 years but whose identify has remained a mystery until now. While there have been a number of proteins described as being candidates for the acute regulator, unequivocal evidence that any of these proteins performed this function had been lacking. Recently, we purified., cloned and expressed a novel, tropic hormone-induced mitochondrial protein which we had earlier hypothesized to be involved in this regulation. Observations described in this proposal indicate that this protein, which we named the Steroidogenic Acute Regulatory (StAR) protein, is the long sought regulatory protein for acute steroidogenesis. Since we have been successful in characterizing the role of StAR in the acute regulation of steroid production, we would now propose to extend this finding into what can considered to be one of the most obvious directions to take this work, namely, the determination of the mechanism of action whereby StAR is able to effect the transfer of cholesterol from cellular stores and the outer mitochondrial membrane to the inner mitochondrial membrane and the CSCC. Specifically, we propose the following Aims: 1). to characterize the mitochondrial membrane receptor for StAR and determine if it is similar to other mitochondrial protein precursor receptors 2). to determine the cholesterol binding capacity of StAR using filter binding assays as well as steady state polyacrylamide gel assays. 3). to characterize the mitochondrial "contact sites" in hormone stimulated MA-10 cells and non- stimulated R2C cells in order to determine if StAR and other components of the mitochondrial steroidogenic complex are present at such sites. 4). to construct NH2-terminal mutants for the StAR protein and determine the effects on both mitochondrial import and processing of the protein and on steroidogenesis 5). to construct COOH-terminal deletion mutants for the StAR protein and determine the effects on both mitochondrial import and steroidogenesis. 6). to determine the role of phosphorylation on the activity of StAR by using MA-10 Leydig tumor cells and y 1 adrenal tumor cells. (7). to construct an expression system designed to produce large quantities of the StAR precursor to be used in in vitro systems in order to more specifically demonstrate StAR function and mechanism of action. These Aims will be performed using standard methodology and their successful completion will add interesting new information concerning the mechanism of action of a novel, hormone induced protein which is specific to steroidogenic tissue. These studies are important since StAR occupies such a fundamental and indispensable role in what can arguably be called one of the most important functions in the body, namely the production of mineralocorticoids, glucocorticoids and gonadal steroids. Indeed, as dramatically pointed out in recent studies, absence of functional StAR results in the condition of lipoid Congenital Adrenal hyperplasia, a lethal syndrome caused by mutations in the StAR gene. In summary, it is our intent to continue to study the characteristics of this novel regulatory protein with the goal of determining the mechanism whereby cholesterol is transferred to the CSCC.